Approximately 40 human chemokines have been described, that function, at least in part, by modulating a complex and overlapping set of biological activities important for the movement of lymphoid cells and extravasation and tissue infiltration of leukocytes in response to inciting agents (See, for example: Ponath, P., Exp. Opin. Invest. Drugs (1998) 7:1-18). These chemotactic cytokines, or chemokines, constitute a family of proteins, approximately 8-10 kDa in size. Chemokines appear to share a common structural motif, that consists of 4 conserved cysteines involved in maintaining tertiary structure. There are two major subfamilies of chemokines: the “CC” or β-chemokines and the “CXC” or α-chemokines. The receptors of these chemokines are classified based upon the chemokine that constitutes the receptor's natural ligand. Receptors of the β-chemokines are designated “CCR”; while those of the α-chemokines are designated “CXCR.”
Chemokines are considered to be principal mediators in the initiation and maintenance of inflammation (see Chemokines in Disease published by Humana Press (1999), Edited by C. Herbert; Murdoch, et al., Blood (2000) 95:3032-3043). More specifically, chemokines have been found to play an important role in the regulation of endothelial cell function, including proliferation, migration and differentiation during angiogenesis and re-endothelialization after injury (Gupta, et al., J. Biolog. Chem. (1998) 7:4282-4287). Two specific chemokines have been implicated in the etiology of infection by human immunodeficiency virus (HIV).
In most instances, HIV initially binds via its gp120 envelope protein to the CD4 receptor of the target cell. A conformational change appears to take place in the gp120 which results in its subsequent binding to a chemokine receptor, such as CCR5 (Wyatt, et al., Science (1998) 280:1884-1888). HIV-1 isolates arising subsequently in the infection bind to the CXCR4 chemokine receptor. In view of the fact that the feline immunodeficiency virus, another related retrovirus, binds to a chemokine receptor without needing to bind first to the CD4 receptor, suggests that chemokine receptors may be the primordial obligate receptors for immunodeficiency retroviruses.
Following the initial binding by HIV to CD4, virus-cell fusion results, which is mediated by members of the chemokine receptor family, with different members serving as fusion cofactors for macrophage-tropic (M-tropic) and T cell line-tropic (T-tropic) isolates of HIV-1 (Carroll, et al., Science (1997) 276:273-276; Feng, et al., Science (1996) 272:872-877; Bleul, et al., Nature (1996) 382:829-833; Oberlin, et al., Nature (1996) 382:833-835; Cocchi, et al., Science (1995) 270:1811-1815; Dragic, et al., Nature (1996) 381:667-673; Deng. et al., Nature (1996) 381:661-666; Alkhatib, et al., Science (1996)272:1955-1958). During the course of infection within a patient, it appears that a majority of HIV particles shift from the M-tropic to the more aggressive pathogenic T-tropic viral phenotype (Miedema, et al., Immune. Rev. (1994) 140:35; Blaak, et al., Proc. Natl. Acad. Sci. (2000) 97:1269-1274; Simmonds, et al. J. Virol. (1996) 70:8355-8360; Tersmette, et al., J. Virol. (1988) 62:2026-2032; Connor, R. I., Ho, D. D., J. Virol. (1994) 68:4400-4408; Schuitemaker, et al., J. Virol. (1992) 66:1354-1360). The M-tropic viral phenotype correlates with the virus's ability to enter the cell following binding of the CCR5 receptor, while the T-tropic viral phenotype correlates with viral entry into the cell following binding and membrane fusion with the CXCR4 receptor. Clinically observations suggest that patients who possess genetic mutations in the CCR5 or CXCR4 appear resistant or less susceptible to HIV infection (Liu, et al., Cell (1996) 86:367-377; Samson, et al., Nature (1996) 382:722-725; Michael, et al., Nature Med. (1997) 3:338-340; Michael, et al., J. Virol. (1998) 72:6040-6047; Obrien, et al., Lancet (1997) 349:1219; Zhang, et al., AIDS Res. Hum. Retroviruses (1997) 13:1357-1366; Rana, et al., J. Virol. (1997) 71:3219-3227; Theodorou, et al., Lancet (1997) 349:1219-1220). Despite the number of chemokine receptors which have been reported to HIV mediate entry into cells, CCR5 and CXCR4 appear to be the only physiologically relevant coreceptors used by a wide variety of primary clinical HIV-1 strains (Zhang, et al., J. Virol. (1998) 72:9307-9312; Zhang, et al., J. Virol. (1999) 73:3443-3448; Simmonds, et al., J. Virol. (1988) 72:8453-8457). Fusion and entry of T-tropic viruses that use CXCR4 are inhibited by the natural CXC-chemokine stromal cell-derived factor-1, whereas fusion and entry of M-tropic viruses that use CCR5 are inhibited by the natural CC-chemokines namely, Regulated on Activation Normal T-cell Expressed and Secreted (RANTES) and Macrophage Inflammatory proteins (MIP-1 alpha and beta).
However, the binding of chemokine receptors to their natural ligands appears to serve a more evolutionary and central role than only as mediators of HIV infection. The binding of the natural ligand, pre-B-cell growth-stimulating factor/stromal cell derived factor (PBSF/SDF-1) to the CXCR4 chemokine receptor provides an important signaling mechanism: CXCR4 or SDF-1 knock-out mice exhibit cerebellar, cardiac and gastrointestinal tract abnormalities and die in utero (Zou, et al., Nature (1998) 393:591-594; Tachibana, et al., Nature (1998)393:591-594; Nagasawa, etal., Nature (1996)382:635-638). CXCR4-deficient mice also display hematopoietic defects (Nagasawa, et al., Nature (1996) 382:635-638); the migration of CXCR4 expressing leukocytes and hematopoietic progenitors to SDF-1 appears to be important for maintaining B-cell lineage and localization of CD34+ progenitor cells in bone marrow (Bleul, et al., J. Exp. Med. (1998) 187:753-762; Viardot, et al., Ann. Hematol. (1998) 77:195-197; Auiti, et al., J. Exp. Med. (1997) 185:111-120; Peled, et al., Science (1999) 283:845-848; Qing, et al., Immunity (1999) 10:463-471; Lataillade, et al., Blood (1999) 95:756-768; Ishii, et al., J. Immunol. (1999) 163:3612-3620; Maekawa, et al., Internal Medicine (2000) 39:90-100; Fedyk, et al., J. Leukocyte Biol. (1999) 66:667-673; Peled, et al., Blood (2000) 95:3289-3296).
Blood cells play a crucial part in maintaining the health and viability of animals, including humans. White blood cells include neutrophils, macrophage, eosinophils and basophils/mast cells as well the B and T cells of the immune system. White blood cells are continuously replaced via the hematopoietic system, by the action of colony stimulating factors (CSF) and various cytokines, in particular on stem cells and progenitor cells in hematopoietic tissues. The nucleotide sequences encoding a number of these growth factors have been cloned and sequenced. Perhaps the most widely known of these is granulocyte colony stimulating factor (G-CSF) which has been approved for use in counteracting the negative effects of chemotherapy by stimulating the production of white blood cells and progenitor cells (peripheral blood stem cell mobilization). A discussion of the hematopoietic effects of this factor can be found, for example, in U.S. Pat. No. 5,582,823, incorporated herein by reference.
Several other factors have been reported to increase white blood cells and progenitor cells in both human and animal subjects. These agents include granulocyte-macrophage colony stimulating factor (GM-CSF), Interleukin-1 (IL-1), Interleukin-3 (IL-3), Interleukin-8 (IL-8), PIXY-321 (GM-CSF/IL-3 fusion protein), macrophage inflammatory protein, stem cell factor, thrombopoietin and growth related oncogene, as single agents or in combination (Dale, D., et al., Am. J. of Hematol. (1998) 57:7-15; Rosenfeld, C., et al., Bone Marrow Transplantation (1997) 17:179-183; Pruijt, J., et al., Cur. Op. in Hematol. (1999) 6:152-158; Broxmeyer, H., et al., Exp. Hematol. (1995) 23:335-340; Broxmeyer, et al., Blood Cells, Molecules and Diseases (1998) 24:14-30; Glaspy, J., et al., Cancer Chemother. Pharmacol. (1996) 38 (suppl): S53-S57; Vadhan-Raj, S., et al., Ann. Intern. Med (1997) 126:673-681; King, A., et al., Blood (2001) 97:1534-1542; Glaspy, J., et al., Blood (1997) 90:2939-2951).
While endogenous growth factors are pharmacologically effective, the well known disadvantages of employing proteins and peptides as pharmaceuticals underlies the need to add to the repertoire of such growth factors with agents that are small molecules. In another aspect, such small molecules are advantageous over proteins and peptides where production in large quantities are desired.
The signal provided by SDF-1 on binding to CXCR4 may also play an important role in tumor cell proliferation and regulation of angiogenesis associated with tumor growth (See “Chemokines and Cancer” published by Humana Press (1999), Edited by B. J. Rollins; Arenburg, et al., J. Leukocyte Biol. (1997) 62:554-562; Moore, et al., J. Invest. Med. (1998) 46:113-120; Moore, et al., Trends Cardiovasc. Med (1998) 8:51-58; Seghal, et al., J. Surg. Oncol. (1998) 69:99-104); the known angiogenic growth factors VEG-F and bFGF, up-regulate levels of CXCR4 in endothelial cells, and SDF-1 can induce neovascularization in vivo (Salcedo, et al., Am. J. Pathol. (1999) 154:1125-1135); leukemia cells that express CXCR4 migrate and adhere to lymph nodes and bone marrow stromal cells that express SDF-1 (Burger, et al., Blood (1999) 94:3658-3667; Arai, et al., Eur. J. Haematol. (2000) 64:323-332; Bradstock, et al., Leukemia (2000) 14:882-888).
The binding of SDF-1 to CXCR4 has also been implicated in the pathogenesis of atherosclerosis (Abi-Younes, et al., Circ. Res. (2000) 86:131-138), renal allograft rejection (Eitner, et al., Transplantation (1998) 66:1551-1557), asthma and allergic airway inflammation (Yssel, et al., Clinical and Experimental Allergy (1998) 28:104-109; J. Immunol. (2000) 164:5935-5943; Gonzalo, et al., J. Immunol. (2000) 165:499-508), Alzheimer's disease (Xia, et al., J. Neurovirology (1999) 5:32-41) and arthritis (Nanki, et al., J. Immunol. (2000) 164:5010-5014).
In attempting to better understand the relationship between chemokines and their receptors, recent experiments to block the fusion, entry and replication of HIV via the CXCR4 chemokine receptor were carried out through the use of monoclonal antibodies or small molecules that appear to suggest a useful therapeutic strategy (Schols, et al., J. Exp. Med. (1997) 186:1383-1388; Schols, et al., Antiviral Research (1997) 35:147-156; Bridger, et al., J. Med. Chem. (1999) 42:3971-3981; Bridger, et al., “Bicyclam Derivatives as HIV Inhibitors” in Advances in Antiviral Drug Design (1999) Volume 3:161-229, published by JAI press, Edited by E. De Clercq). Small molecules, such as bicyclams, appear to specifically bind to CXCR4 and not CCR5 (Donzella, et al., Nature Medicine (1998) 4:72-77). These experiments demonstrated interference with HIV entry and membrane fusion into the target cell in vitro. More recently, bicyclams were also shown to inhibit fusion and replication of Feline Immunodeficiency Virus (FIV) that uses CXCR4 for entry (Egberink, et al., J. Virol. (1999) 73:6346-6352).
Additional experiments have shown that the bicyclam dose-dependently inhibits binding of 125I-labeled SDF-1 to CXCR4 and the signal transduction (indicated by an increase in intracellular calcium) in response to SDF-1. Thus, the bicyclam also functioned as an antagonist to the signal transduction resulting from the binding of stromal derived factor or SDF-1α, the natural chemokine to CXCR4. Bicyclams also inhibited HIV gp120 (envelope)-induced apoptosis in non-HIV infected cells (Blanco, et al., Antimicrobial Agents and Chemother. (2000) 44:51-56).
U.S. Pat. Nos. 5,583,131; 5,698,546; 5,817,807; 5,021,409; and 6,001,826, which are incorporated herein in their entirety by reference, disclose cyclic compounds that are active against HIV-1 and HIV-2 in in vitro tests. It was subsequently discovered and further disclosed in PCT WO 02/34745 that these compounds exhibit anti-HIV activity by binding to the chemokine receptor CXCR4 expressed on the surface of certain cells of the immune system. This competitive binding thereby protects these target cells from infection by HIV which utilize the CXCR4 receptor for entry. In addition, these compounds antagonize the binding, signaling and chemotactic effects of the natural ligand for CXCR4, the chemokine stromal cell-derived factor 1α (SDF-1). We further disclosed that these novel compounds demonstrate protective effects against HIV infection of target cells by binding in vitro to the CCR5 receptor.
Additionally we have disclosed in U.S. Pat. No. 6,365,583 that these cyclic polyamine antiviral agents described in the above-mentioned patents/patent applications have the effect of enhancing production of white blood cells as well as exhibiting antiviral properties. Thus, these agents are useful for controlling the side-effects of chemotherapy, enhancing the success of bone marrow transplantation, enhancing wound healing and burn treatment, as well as combating bacterial infections in leukemia.
More recently, we disclosed in PCT WO 00/56729, PCT WO 02/22600, PCT WO 02/22599, and PCT WO 02/34745 a series of heterocyclic compounds that exhibit anti-HIV activity by binding to the chemokine receptors CXCR4 and CCR5 expressed on the surface of certain cells of the immune system. This competitive binding thereby protects these target cells from infection by HIV which utilize the CXCR4 or CCR5 receptors for entry. In addition, these compounds antagonize the binding, signaling and chemotactic effects of the natural ligand for CXCR4, the chemokine stromal cell-derived factor 1α (SDF-1) and/or the natural ligand for CCR5, the chemokine RANTES.
The chemokine receptor, CXCR4 has been found to be essential for the vascularization of the gastrointestinal tract (Tachibana, et al., Nature (1998) 393:591-594) as well as hematopoietic and cerebellar development (Zou, et al., Nature (1998) 393:591-594). Interference with any of these important functions served by the binding of pre-B-cell growth-stimulating factor/stromal derived factor (PBSF/SDF-1) to the CXCR4 chemokine receptor results in lethal deficiencies in vascular development, hematopoietic and cardiogenesis. Similarly, fetal cerebellar development appears to rely upon the effective functioning of CXCR4 in neuronal cell migration and patterning in the central nervous system. This G-protein-coupled chemokine receptor appears to play a critical role in ensuring the necessary patterns of migration of granule cells in the cerebellar anlage.
Herein, we disclose compounds that have unique chemical attributes and that exhibit protective effects against HIV infection of target cells by binding to chemokine receptor CXCR4 or CCR5 in a similar manner to the previously disclosed macrocyclic compounds. In addition, these compounds antagonize the binding, signaling and chemotactic effects of the natural ligand for CXCR4, the chemokine stromal cell-derived factor 1α (SDF-1) and/or the natural ligand for CCR5 (the chemokine RANTES).
Further, the compounds of the invention have the effect of increasing progenitor cells and/or stem cells. Even further, the compounds have the effect of enhancing production of white blood cells as well as exhibiting antiviral properties. Thus, these agents are useful where treatment affects the activities within the bone marrow resulting in leukopenia, thus controlling the side-effects of chemotherapy, radiotherapy, enhancing the success of bone marrow transplantation, enhancing wound healing and burn treatment, as well as combating bacterial infections in leukemia. Further, the compounds of the invention effect regeneration of cardiac tissue.
Citation of the above documents is not intended as an admission that any of the foregoing is pertinent prior art. All statements as to the date or representation as to the contents of these documents is based on the information available to the applicants and does not constitute any admission as to the correctness of the dates or contents of these documents. Further, all documents referred to throughout this application are hereby incorporated in their entirety by reference herein.